Project Summary/Abstract Voltage-gated Na+ channels (Nav) are essential for normal atrial excitability and function, as evidenced by the strong link between dysfunction in the primary cardiac Nav alpha subunit (Nav1.5) and atrial arrhythmias. In particular, congenital or acquired defects in Nav that promote inappropriate ?late? Na+ current (INa,L) commonly result in atrial as well as ventricular arrhythmia. While increased late INa has been linked to atrial fibrillation (AF) in patients and in animal disease models, little is known about the underlying pathways for dysregulation. Moreover, the potential of INa,L as a therapeutic target in AF, while viewed with optimism, remains controversial and difficult to assess due to nonspecific nature of pharmacological agents and limitations of available animal models. Nav1.5 is tightly regulated within local signaling domains that control channel post-translational modification. Nav1.5 phosphorylation is an important pathway for modulating channel function and level of INa,L. In ventricle, changes in Nav1.5 phosphorylation have been linked by our group and others to arrhythmogenic INa,L. In contrast, the roles for Nav1.5 phosphorylation in atria are unknown and essentially unstudied. In fact, despite its clear role in atrial function and atrial arrhythmias we know essentially nothing regarding the molecular mechanisms that control atrial INa phosphoregulation. Perhaps more surprising, almost nothing is known about the pathways underlying Nav1.5 dephosphorylation, in either atria or ventricle. We have identified a novel pathway for specific regulation of atrial INa,L by CaMKII with an important role in arrhythmogenesis. Our preliminary data indicate that this pathway includes a previously unappreciated negative regulatory axis for Nav1.5 mediated by protein phosphatase 2A (PP2A). Furthermore, our new data support that PP2A-dependent antagonism of CaMKII phosphorylation occurs within a macromolecular complex organized by ankyrin-G. Finally, we provide initial evidence that this regulatory pathway is altered in animal models and human AF. Our long-term goal is to define the molecular pathway for CaMKII-dependent phosphoregulation of Nav1.5 in atrial myocytes, to understand the role of this pathway in AF, and test whether it may be manipulated for therapeutic benefit. Our central hypothesis is that dysfunction in the PP2A- dependent regulatory axis exacerbates imbalance in Nav1.5 phosphoregulation induced by CaMKII hyperactivity, leading to increased INa,L and ultimately increased AF susceptibility downstream of defects in Ca2+ homeostasis. We will: 1) Define the molecular pathway controlling CaMKII-dependent phosphorylation of atrial Nav1.5; 2) Determine the role of atrial INa phosphoregulation in modulating atrial Ca2+ homeostasis, excitability, and function; and 3) Determine the impact of Nav1.5 phosphoregulation in AF susceptibility and progression. We assert that a fundamental understanding of atrial INa regulation and downstream function is essential for both our understanding of AF mechanisms and the rational design of new therapies.